Transcriptional regulation of human CYP27 integrates retinoid, peroxisome proliferator-activated receptor, and liver X receptor signaling in macrophages

Abstract

Cholesterol uptake and efflux are key metabolic processes associated with macrophage physiology and atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha) have been linked to the regulation of these processes. It remains to be identified how activation of these receptors is connected and regulated by endogenous lipid molecules. We identified CYP27, a p450 enzyme, as a link between retinoid, PPARgamma, and LXR signaling. We show that the human CYP27 gene is under coupled regulation by retinoids and ligands of PPARs via a PPAR-retinoic acid receptor response element in its promoter. Induction of the enzyme's expression results in an increased level of 27-hydroxycholesterol and upregulation of LXR-mediated processes. Upregulated CYP27 activity also leads to LXR-independent elimination of CYP27 metabolites as an alternative means of cholesterol efflux. Moreover, human macrophage-rich atherosclerotic lesions have an increased level of retinoid-, PPARgamma-, and LXR-regulated gene expression and also enhanced CYP27 levels. Our findings suggest that nuclear receptor-regulated CYP27 expression is likely to be a key integrator of retinoic acid receptor-PPARgamma-LXR signaling, relying on natural ligands and contributing to lipid metabolism in macrophages.

FIG. 1.

Regulated expression of CYP27 by retinoids and PPARγ activators. Total RNAs isolated from primary human monocytes (Mo) and macrophages (MΦ) or MonoMac-6 cells after 2 days in culture were used for real-time quantitative RT-PCR analysis as described in Materials and Methods. Absolute molecule numbers of PPARγ (A), LXRα (B), and CYP27 (C) were determined and normalized to cyclophilin levels. Every transcript was measured in triplicates. Primary human monocytes were treated with Rosiglitazone (D, E, and F) or MonoMac-6 cells (G, H, and I) and cultured in the presence of the indicated ligands or vehicle (ATRA. 1 μM; 9-cis-RA, 1 μM; AM580 [RARα agonist], 100 nM; LG268 [RXR agonist], 100 nM; WY14643 [PPARα agonist], 10 μM; Rosiglitazone [PPARγ agonist], 1 μM; GW501516 [PPARδ agonist], 1 μM; T0901317 [LXRα agonist], 1 μM). (D, E, F, G, and I) After 2 days, total RNA was isolated and real-time quantitative RT-PCR analysis was performed. The means of at least three independent measurements ± SD are presented. (H) Northern analysis was performed on total RNA isolated from MonoMac-6 cells as described in Materials and Methods. Membranes were probed with labeled CYP27 and 36B4 cDNA probe. *, P < 0.01; **, P < 0.05 (compared to the respective control value).

FIG. 2.

Regulation of CYP27 is dose and time dependent. (A) Northern analysis was performed on RNA isolated from retinoid and PPARγ agonist-treated MonoMac-6 cells. (B) Real-time quantitative PCR analysis, showing a detailed time course of CYP27 induction, on RNA isolated from MonoMac-6 cells treated as indicated. Cyclophilin D was used as a normalizer during quantitative PCR, unless indicated otherwise. (C) ATRA was used to show the dose dependence of CYP27 induction in MonoMac-6 cells. (D and E) MonoMac-6 cells were cultured in the indicated serum-containing medium for 2 days, and CYP27 (D) and transglutaminase (Tg) (E) mRNA levels (bars) were determined by quantitative RT-PCR. The ATRA concentrations (D) in the various sera were measured by HPLC as described in Materials and Methods. CCSS, charcoal-stripped medium; LpDS, lipoprotein-deficient medium. (F and G) MonoMac-6 cells cultured in fetal calf serum (FCS) were treated with the RAR inverse agonist AGN193109, and CYP27 (F) and transglutaminase (G) mRNA levels were determined by quantitative PCR and compared to those in AIMV. The means of at least three determinations ± SD are shown. *, P < 0.01; **, P < 0.05 (compared to the respective control value).

FIG. 3.

The promoter of the human CYP27 gene is a direct target for complex regulation by RAR-RXR and PPARγ-RXR heterodimers. (A and B) CV-1 cells were cotransfected with hCYP27-853-pCAT, hCYP27-649-pCAT, hCYP27-217-pCAT, the indicated receptors, and β-galactosidase and treated with the receptor agonists 9-cis-RA (1 μM), Rosiglitazone (1 μM), both, or vehicle for 48 h. Cells were lysed, and the CAT level and β-galactosidase activity were assayed as described in Materials and Methods. CAT levels were normalized to β-galactosidase activity. All experiments were done in triplicates. The means of at least three determinations ± SD are shown. (C) Chromatin immunoprecipitation was performed with anti-RAR, anti-RXR, and anti-PPARγ antibodies (ab), and the DNA content was determined with two quantitative PCR assays specific for PRRE-B. DNA precipitated with anti-RXR and anti-PPARγ antibodies was analyzed by a human LXRα PPAR response element (PPRE)-specific assay. The results are shown as percentages of input DNA. All measurements were done in triplicate. All chromatin results were verified from independent chromatin preparations. *, P < 0.01; **, P < 0.05 (compared to the respective control [C] value).

FIG. 4.

Identification of RXR-RAR and RXR-PPARγ binding sites in the human CYP27 promoter. (A) Electrophoretic mobility shift analysis for hCYP27-PRRE-B was performed by using in vitro-translated receptors of mPPARγ and hRXRα (left panel) and hRARα and hRXRα (right panel) and α³²P-labeled oligonucleotides in the absence or presence of the indicated ligands (Rosiglitazone [1 μM] or 9-cis-RA [1 μM]) as described in Materials and Methods. For competition experiments, cold hCYP27-PRRE-B (self [S]), consensus DR1, or DR5 was used at ×10 and ×20 concentrations. For supershift experiments, the receptors were preincubated with the indicated antibodies prior to the binding reaction. (B) The response element was analyzed in a cell-based binding assay. The indicated VP fusion nuclear receptors were cotransfected with the response element containing a luciferase reporter plasmid, and the normalized reporter activity ± SD are shown. (C) Two copies of hCYP27-PRRE-B and half-site mutants were cloned upstream from the TK-Luc reporter. They were cotransfected into CV-1 cells with the indicated receptors and β-galactosidase and treated with the indicated ligands: AM580 (100 nM), Rosiglitazone (1 μM), LG268 (100 nM), or vehicle. Luciferase activity was normalized to β-galactosidase activity. *, P < 0.01 compared to the respective control value.

FIG. 5.

Induction of CYP27 is accompanied by increased enzyme activity that leads to production of 27-hydroxycholesterol. (A) MonoMac-6 cells were treated with water-soluble cholesterol (100 μg/ml) and the indicated ligands for 2 days in RPMI supplemented with insulin-transferrin-sodium selenite medium supplement. 27-Hydroxyxholesterol and 3β-hydroxy-5-cholestenoic acid contents in the cell pellet were determined and normalized to cell number as described in Materials and Methods. The inset shows Western blot analysis of CYP27 protein levels in MonoMac-6 cells treated with ATRA (1 μM), 9-cis-RA (1 μM), or vehicle (for details, see Materials and Methods). (B) As for panel A, MonoMac-6 cells were cholesterol loaded and treated with ligands or vehicle as indicated, and 27-hydroxycholesterol was determined. (C) MonoMac-6 cells were treated with increasing amounts of 27-hydroxycholesterol, and a dose-response curve was determined. To analyze synergy, cells were treated with ATRA (1 μM), 9-cis-RA (1 μM), AM580 (100 nM), or LG268 (100 nM) and 27-hydroxycholesterol simultaneously. Transcript levels of ABCA1 were measured by real-time quantitative RT PCR and normalized to cyclophilin levels. The results are shown on a logarithmic scale. (D) CV-1 cells were cotransfected with Gal-hLXRα-LBD, hRXRα-LDB, pMH100-TK-Luc, and β-galactosidase plasmids and treated with LXR activators 25-hydroxycholesterol, 27-hydroxycholesterol, and 24(S)25-epoxycholesterol at the indicated concentrations in the absence or presence of 9-cis-RA (1 μM). Luciferase activity was normalized to β-galactosidase activity, and EC₅₀s values were calculated from the dose-response curves (inset). *, P < 0.01 compared to the respective control value.

FIG. 6.

Retinoid-induced CYP27 promotes an LXR response. Primary human monocytes were isolated and treated with the indicated retinoids. Total RNA was isolated, and ABCA1 (A) and ABCG1 (B) mRNA levels were determined by real-time quantitative RT-PCR. (C and D) Primary human fibroblasts isolated from healthy adults and patients with CTX (CYP27^−/−) were treated with the indicated ligands for 2 days. Total RNA was isolated, and CYP27 (C) and ABCA1 (D) mRNA levels were determined by real-time quantitative RT-PCR. *, P < 0.01; **, P < 0.05 (compared to the respective control value).

FIG. 7.

Elements of the retinoid-PPARγ-CYP27-LXRα pathway are present in human macrophage-rich atherosclerotic lesions. (A) A human femoral artery containing a macrophage-rich fatty lesion was dissected, and total RNA was extracted from the indicated parts. 1, normal intima; 2, normal media and adventitia; 3, intima of atherosclerotic lesion; 4, media and adventitia of atherosclerotic lesion. (B to G) Transcript levels of the indicated genes were measured by real-time PCR and normalized to cyclophilin levels. Three similar lesions from various individuals were analyzed with essentially the same results. Results from a representative experiment are presented. *, P < 0.01 compared to the respective control value. (H) Integration of retinoid, PPAR, and LXR signaling via CYP27, leading to enhanced LXR-dependent and -independent cholesterol efflux. TZD, thiazolidinedione.